The cardiac neural crest is a unique population of cells that undergo extensive migration through the embryo and contribute to many aspects of cardiac development, including the formation of the interventricular septum, the partitioning of the cardiac outflow tract, and the development of the heart valves. Defects in cardiac neural crest are at the root of many congenital heart defects found in live births, and these issues often require surgical repair. Despite this clear importance in human development, our understanding of cardiac neural crest biology remains incomplete. Here we propose to determine the role of two transcription factors, MafB and Krox20, in the development of the cardiac neural crest. Interestingly, these two factors are expressed in the migrating cardiac neural crest, and not in other migrating neural crest populations, suggesting that they play specific roles in cardiac neural crest development. As transcription factors, MafB and Krox20 function to regulate expression of additional genes, and thereby they can have broad impacts on the behavior of the cardiac neural crest cells. By eliminating MafB and Krox20 function in the chick cardiac neural crest we will identify any resulting defects. We hypothesize that loss of MafB or Krox20 will result in cardiac defects that closely resemble many congenital heart defects, and thereby demonstrate the importance of these gene products for normal cardiac neural crest development. To achieve these goals, we will examine not only cardiac morphology, but also the migratory path taken by the cardiac neural crest cells, as well as the expression of target downstream genes through candidate-based and systems-level experiments. We will also identify important transcriptional inputs into MafB and Krox20 expression. Following these experiments, we will assemble a gene regulatory network model that describes the cascade of gene expression upstream and downstream of MafB and Krox20 in the cardiac neural crest, and provides the necessary framework to determine what makes the cardiac neural crest unique from other neural crest linages. This knowledge will inform future studies in which other neural crest cells will be reprogrammed to compensate for loss of cardiac neural crest, and thereby rescue cardiac neural crest-related congenital heart defects.